Biocompatible material and process for manufacturing the same

ABSTRACT

In order to provide a biocompatible material consisting mainly of a fish scale-derived hydroxyapatite and a fish scale-derived collagen, which is adjusted so that easily digested and absorbed in a human body, the material contains a composite consisting mainly of a fish scale-derived hydroxyapatite and a fish scale-derived collagen.

FIELD OF THE INVENTION

The present invention relates to a biocompatible material using fishscales, which is so prepared that it can be easily absorbed to organismssuch as mammals, and to a process for manufacturing the material.

BACKGROUND ART

Calcium is an important component which forms human bones and tooth. Itis further known that the substance is essential also in thecardiovascular and neurotransmission. Furthermore, it is well-known thatcollagen exists in almost all tissues including human bones, cartilages,tooth, skin, vessels and organs, and in other words, it is an essentialcomponent which is indispensable for organization of human body.However, due to decrease of the calcium and collagen for variousreasons, so-called aging symptoms such as osteoporosis andhypometabolism become obvious.

Hydroxyapatite is a common designation for inorganic chemicalsconsisting mainly of calcium and phosphoric acids and expressed by aformula Ca₁₀(PO₄)₆(OH)₂. It is a main component of bones and tooth.Concerning a process for manufacturing, the hydroxyapatite the one usingfish scale has been proposed. As a prior art document which describesthe process for manufacturing the fish scale-derived hydroxyapatite,there is a disclosure in JP Laid-Open 2001-211895.

On the other hand, collagen belongs to proteins and is often extractedfrom collagen tissues of mammals like a cow and a pig. However, thesemammals are always facing a risk of infection of pathogens such asprions which are eliminated with difficulties using ordinarysterilization and pasteurization methods, thus a manufacture of collagenfrom fish which are free from such fear. A fish skin is used in themanufacture of collagen from fish, a problem is raised that the collagenfrom the fish skin smells like fish and generates white turbidityleading to a low permeability. Therefore, several processes formanufacturing collagen from fish scale, wherein the processes cancontribute to a solution for fish-like smells particular to fish, havebeen proposed. As prior art documents which describe the process formanufacturing collagen from the fish scale, there are disclosures in JPLaid-Open Patent Publications H05-155900, 2001-327599 and 2003-238598.

It is known that the hydroxyapatite as above described or thetraditional calcium are, though poorly absorbed by a human body,biocompatible, and further, once absorbed by a human body, thesesubstances can supplement these components that are deficient inside ahuman body. Starting from this common knowledge, the inventors havedevotedly studied in order to facilitate a digestion and absorption by ahuman body, so that they have finally found out that, although calciumhas conventionally been considered as showing a poor absorption, thecollagen is assigned a scaffolding function by forming a composite fromthe hydroxyapatite and collagen, so that the hydroxyapatite absorbed onthe collagen is well absorbed by a human body. Based on these findings,the inventors have then elaborated the present invention.

SUMMARY OF THE INVENTION

The present invention has an object to provide a biocompatible materialconsisting mainly of a fish scale-derived hydroxyapatite and a fishscale-derived collagen which material is so prepared that it is easilydigested and absorbed by a human body.

The present invention has further an object to provide a process formanufacturing a biocompatible material consisting mainly of a fishscale-derived hydroxyapatite and a fish scale-derived collagen whichmaterial is digested and absorbed by a human body in a manner as easy aspossible.

To achieve the above-mentioned object, this invention is characterizedin that it contains a composite consisting mainly of a hydroxyapatitederived from fish scales and a collagen derived from fish scales aswell.

In addition, this invention is characterized in that the compositecontains a hydroxyapatite derived from fish scales and a collagenderived from fish scales as well, in the weight percent ratio of about8:2.

Additionally, this invention is characterized in that the compositecontains 4.2 g of water, 20.4 g of collagen, 390 mg of sodium, 14.2 g ofphosphorus, 70.2 g of hydroxyapatite and 323 mg of magnesium per 100 g.

Moreover, this invention is characterized in that the hydroxyapatite hasa molecular weight of 1,000.

Furthermore, this invention is characterized in that the collagen has amolecular weight of 500 to 1,000.

Further, this invention is characterized in that the hydroxyapatitecontains 33.5 g of calcium, 17.2 g of phosphorus, 390 mg of magnesium,900 mg of sodium, 0.1 g or less of protein and 3.4 g of water per 100 g.

Still further, this invention is characterized in that the collagencontains 4.5 g of water, 0.2 g of lipids, 0.2 g of ash and 52.6 mg ofsodium per 100 g.

Still further, this invention is characterized in that in obtaining theabove-mentioned composite, an extract of a fish scale-derivedhydroxyapatite (with water content of 70 to 75% by weight) and the oneof a fish scale-derived collagen (with water content of 40 to 60% byweight) are mixed in the weight percent ratio of about 8:2 and thenstirred, and after that the mixture is dried by hot blast in order toobtain the composite.

Still further, this invention is characterized in that a production ofthe composite is incorporated in the process for producing the fishscale-derived hydroxyapatite.

Still further, this invention is characterized in that a production ofthe composite is incorporated in the process for producing the fishscale-derived collagen.

As above described, in a biocompatible material according to the presentinvention it is characterized in that the material is safe as it is madeof a fish scale, and that the composite is formed by mixing respectiveextracts of the hydroxyapatite and of the collagen, stirring anddepositing the mixture, so that the collagen is absorbed and bonded ingaps between crystal structures of hydroxyapatite, which assigns ascaffolding function to the collagen contained in the composite and moreeasily realizes an absorption by a human body of the hydroxyapatiteabsorbed on the collagen. Further, in view of the present circumstanceswhere the calcium intake of the Japanese falls below the nutritionalallowance and those affected by osteoporosis increase yearly due to aninsufficient calcium intake during the meal and decrease in calciumabsorbing ability accompanied by aging, etc., the present invention iseffective as a solution for the problematic situation. Additionally,following the process for producing according to the present invention,the production of the biocompatible material according to the presentinvention can be incorporated into the process for producing the fishscale derived hydroxyapatite or collagen, so that the production isfacilitated.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flowchart diagram showing an example of process forproducing a hydroxyapatite/collagen composite according to the presentinvention.

FIG. 2 is a flowchart diagram showing another example of process forproducing a hydroxyapatite/collagen composite according to the presentinvention.

FIG. 3 is an image of a marketed hydroxyapatite/collagen mixture as seenusing scanning electron microscope.

FIG. 4 is an image of a composite according to the present invention, asseen using scanning electron microscope.

FIG. 5 is an image of a marketed hydroxyapatite as seen using scanningelectron microscope.

FIG. 6 is an explanatory view showing a femoral site for analysis, whichis isolated from a model mouse.

FIG. 7 is a pQCT image of the site for analysis, in OVX control modelmice and those treated with a composite according to the presentinvention.

FIG. 8 is a 3-D image of trabecular structure in OVX control model miceand sham operated ones, in traverse and a longitudinal cross section.

FIG. 9 is a 3-D image of trabecular structure in model mice treated byrisedronate, MK4, MK7 and a composite according to the presentinvention, in a cross section and a longitudinal section.

DETAILED DESCRIPTION OF THE EXAMPLES

Hereinafter reference is made to the case where a production of abiocompatible material according to the present invention isincorporated into a production process of hydroxyapatite, but it may bealso incorporated into a production process of collagen, which isdescribed below.

FIG. 1 is, as above stated, a flowchart diagram showing an example ofprocess for producing a composite, focused on the production line ofhydroxyapatite. As shown on the Figure, the process for producing thecomposite according to Example 1 comprises a soaking step 1 for soakingdried fish scales in water, and an acid treatment step 2 for subjectingto acid treatment the fish scales which has been previously soaked inwater and then drained, by soaking them in an acidic aqueous solution at15 to 35° C. for 10 to 30 minutes so as to extract hydroxyapatitecontained in the fish scales, and a first solid-liquid separation step 3for separating a hydroxyapatite extract containing solid resultant fromthe acid treatment step 2 into solid and liquid. The liquid separatedduring the first solid-liquid separation step 3 then proceed to a firstfiltration step 5, while the solid (decalcified fish scales) istransferred to a first washing process 23 of a process for producingcollagen, so that collagen can be produced from the solid.

Fish scales are not particularly limited, and their selection may bemade indifferent to fish types, e.g. whether they are of sea fish orfreshwater fish, but includes scales of carp, tilapia, Japaneseseaperch, true sardine, sea bream, salmon, Japanese horse mackerel,etc., and in particular those of carp, tilapia, Japanese seaperch, etc.are preferable in view of hydroxyapatite and collagen content and arelatively greater availability. However, the fish scales used inpractice may be a mixture of various fish scales. Further, the fishscales may be dried or undried, but in particular dried fish scales arepreferable in view of comfort in handling. In using dried fish scales,they are subjected to a soaking (water treatment) step for soaking themin water prior to an acid treatment so that they restore their originalwater content. In the present example dried fish scales are used.

In a soaking step 1 dried fish scales are soaked in water so that theyrestore their original water content as above mentioned, and theoriginal water content is restored e.g. by placing a permeable nylon netfilled with dried fish scales into a tank with water of an amount of 2.5to 4.0 times as much as the dried fish scales, and thus soaking thedried fish scales in water for 10 to 14 hours. A nylon net of mesh sizeof 1 mm or less is preferably selected. The dried fish scales which haveundergone the soaking step 1 are drained and then proceed to asubsequent acid treatment step 2.

In the acid treatment step 2 ash consisting mainly of hydroxyapatite isseparated from dried fish scales. Drained fish scales are removed from anylon net, and then placed into an acidic aqueous solution, so that theabove-mentioned ash content is separated from the above-mentioned fishscales. The acidic aqueous solution is not particularly limited, but maybe an organic acid aqueous solution or a mineral acid aqueous solution,and in particular a hydrochloric acid aqueous solution is preferable. Inthe hydrochloric acid aqueous solution preferred conditions varyaccording to fish scale type, but e.g. the solution is preferablyproduced by pouring a hydrochloric acid of purity of 35% (of 65 wt %with regard to the fish scales, and of a hydrochloric acid concentrationof 5.7%) into water of an amount of 2.5 to 4.0 times as much as the fishscales.

A soaking duration during which fish scales are soaked in an acidicaqueous solution for an acid treatment varies according to fish scaletype, and is preferably e.g. 10 to 30 minutes, and in particularpreferably 15 minutes under a hydrochloric acid concentration of ahydrochloric acid aqueous solution of the preceding paragraph. Duringthe duration the mixture is stirred using a stirrer. If the soakingduration is less than 10 minutes, the fish scales are not sufficientlydecalcified and a yield of hydroxyapatite turns out low. On the otherhand, there is little need to arrange a soaking duration more than 30minutes. Furthermore, a temperature of the acid aqueous solution is notparticularly limited, but e.g. preferably 15 to 30° C., and morepreferably 25° C. (±5° C.). Under the conditions for hydrochloric acidconcentration of the above-described hydrochloric acid aqueous solutionand soaking duration, fish scales are not sufficiently decalcified in atemperature below 15° C. and a yield of hydroxyapatite turns out low,while over 30° C., a yield of collagen turns out low. In conducting theacid treatment, fish scales are preferably stirred while soaked in theacid aqueous solution. A means for stirring is not particularly limited,a spatula, etc. may be selected other than a stirrer. Once fish scaleshave undergone the acid treatment step 2, they are subsequentlytransferred to a first solid-liquid separation step 3.

In the first solid-liquid separation step 3 a solid content of fishscales is separated using a vibrating sieve, etc. from a treated extractsubjected to an acid treatment step 2, wherein the solid contentseparated during this step is transferred to a collagen production step,while the treated extract containing hydroxyapatite (ash) to a firstfiltration step 5, during which the extract is filtrated.

The first filtration step 5 is arranged for removing particulate foreignsubstance from hydroxyapatite extract, and conducted e.g. using a wovenwire product. A mesh scale of the woven wire product is not particularlylimited, but 20-200 is preferably selected. The hydroxyapatite extractfiltrated during this first filtration step 5 is subsequently deodorizedduring a deodorization step 6.

A deodorization treatment during the deodorization step 6 is notparticularly limited, but may be conducted e.g. using activated carbon.In using the activated carbon, the amount equivalent to 1.5% ofhydroxyapatite extract is preferably selected. The deodorizationtreatment may be conducted e.g. using woody or palm-chip activatedcarbon. The hydroxyapatite extract after the deodorization treatment isfurther filtrated during a second filtration step 7.

A method for filtration in the second filtration step 7 is notparticularly limited, but an apparatus for filtration may be also used,wherein diatomaceous earth is placed into a hydroxyapatite extract, anda resultant liquid is stirred and at the same time filtrated through afilter fabrics coated with diatomaceous earth. The acid-treated liquidafter the filtration is subsequently subjected to a treatment forapatite deposition during an apatite deposition step 8.

A treatment for deposition during the apatite deposition step 8 isconducted for depositing hydroxyapatite by adding an alkali to ahydroxyapatite extract. The alkali is not particularly limited, butincludes sodium hydrate, potassium hydrate, etc. The method for addingthe alkali is not particularly limited. When the alkali is added to thehydroxyapatite extract and stirred until pH turns out to be 6 to 9,hydroxyapatite is deposited. The deposited hydroxyapatite issubsequently separated from the liquid in a second solid-liquidseparation step 9. In the meantime, hydroxyapatite is herein depositedby adding an alkali, but an alkali aqueous solution in place of analkali may be added for depositing hydroxyapatite.

In the second solid-liquid separation step 9 a deposited hydroxyapatiteis separated from a remaining liquid, and the means is not particularlylimited provided that the solid can be separated from the liquid, but afilter press, a centrifuge etc. may be used for this purpose.

Hydroxyapatite separated from a liquid during the above-mentioned secondsolid-liquid separation step 9 is subsequently collected into a tank,desalted during a first desalting step 10, and again separated from aliquid a third solid-liquid separation step 11. The resulting product ismixed to a collagen extract after an adjustment in moisture. A manner ofthe above-mentioned first desalting step 10 is not particularly limited,an ion exchanger, etc. may be used during this step for removing sodiumion contained in the liquid, but a desalting treatment may be alsoconducted by washing with water. In desalting through this washing step,production costs can be reduced.

A collagen extract is preferably produced, as above described, based onacid-treated fish scales separated from a liquid during a firstsolid-liquid separation step 3 of a production line of hydroxyapatiteand following production steps to be described.

It is characterized in that water is added to fish scales separated froma liquid during a first solid-liquid separation step 3 which aresubjected to a pH adjustment during a first washing step 23, and fishscales in water comprise the following steps: a heating treatment step24 for extracting collagen contained in the fish scales, a first coolingstep 25 for cooling to a room temperature a heated liquid containing asolid from the heating treatment step 24, a forth solid-liquidseparation step 26 for separating a solid from a collagen extract cooledduring the first cooling step 25, a first disinfecting step 27 forheating and disinfecting the collagen extract separated during the forthsolid-liquid separation step 26, a second cooling step 28 for cooling toa room temperature the collagen extract disinfected under heat duringthe first disinfecting step 27, a second washing step 29 for adjustingpH of the collagen extract cooled during the second cooling step 28, andan enzymatic degradation step 30 for enzymatically degrading thecollagen extract of which pH is adjusted by adding an enzyme. In themeantime, the fish scales separated from a liquid during the forthsolid-liquid separation step 26 are again heated during a reheatingtreatment step 40 in order to extract a collagen, and following a thirdcooling step 41 a collagen extract is again separated during a fifthsolid-liquid separation step 42 and the resultant product is added tothe collagen extract obtained during the above-mentioned forthsolid-liquid separation step 25, so that the fish scales can be consumedwithout waste.

A collagen extract cooled after a heating treatment and then separatedfrom solid is subjected to a disinfecting treatment during a firstdisinfecting step. The disinfecting treatment is not particularlylimited, but a heated liquid may also be heated to 70 to 80° C.,preferably to 75° C. The heated liquid after the disinfecting treatmentis cooled during a second cooling step 28 to a temperature at which anenzyme can be activated, e.g. 60° C., to be described below, and thenits pH is adjusted during a second washing step 29 to 5 to 8, inparticular 6.5 (±0.2). The adjustment in pH is not particularly limited,but may be conducted by adding an alkali, such as sodium hydrate,potassium hydrate, etc. The collagen extract of which pH has beenadjusted during the second washing step 29 is subjected to a treatmentunder a subsequent enzymatic degradation step 30.

The enzymatic degradation step 30 is for adding an enzyme to a collagenextract subjected to a treatment with regard to pH during a secondwashing step 29, so that a molecular weight of collagen can be reduced.The enzymatic degradation treatment is conducted in order to obtain acollagen of a molecular weight of 500 to 3000, more preferably of 500 to2000, and most preferably of 500 to 1000. An enzyme is not particularlylimited provided that the heated liquid can be enzymatically degraded,but includes e.g. an alkaline enzyme derived from Bacillus licheniformis(Genencor Int. Inc., trade name: Protex 6L), etc. An addition amount ofthe enzyme is not particularly limited, but is preferably 0.01 to 0.1%,more preferably 0.08% of the substrate.

A temperature for enzymatic degradation varies according to enzyme, butis preferably 30 to 70° C., more preferably 60° C. A duration forenzymatic degradation varies according to enzyme, but is preferably 3 to10 hours, more preferably 6 to 8 hours, most preferably 8 hours. If theduration for enzymatic degradation is less than 6 hours, a desiredreduction in molecular weight of collagen cannot be sufficientlyrealized, while under a duration for enzymatic degradation more than 10hours the molecular weight of collagen remains unchanged. Furthermore,the enzymatic degradation treatment is preferably conducted withstirring. A method for the stirring is not particularly limited, bute.g. a stirrer, etc. may be selected for this purpose.

In a collagen extract treated during an enzymatic degradation step 30(it may be referred to as enzyme-treated liquid) an enzyme issubsequently deactivated by heating the extract in an enzymedeactivation step 31 at 80 to 85° C., more preferably at 85° C. for 15minutes. A liquid with a deactivated enzyme is cooled following a forthcooling step 32 to e.g. 60° C. Following the cooling treatment, a solidis removed from an enzyme-treated liquid during a third filtration step33. The third filtration step 33 is not particularly limited providedthat the solid can be removed, but e.g. a clarifying filtration etc.using a filter press is preferable. For clarifying filtration afiltration apparatus etc. may be used, in which a diatomaceous earth isplaced into the enzyme-treated liquid, and the resulting liquid isstirred and at the same time filtrated through a filter fabrics coatedwith diatomaceous earth. Furthermore, a deodorizing treatment ispreferably conducted during the third filtration step 33. In thedeodorizing treatment, a woody or palm-chip activated carbon e.g. may beused.

Salt contained in a filtrate after a clarifying filtration (it may bereferred to as treated liquid) is removed from the treated liquid duringa second desalting step 34. The desalting treatment is not particularlylimited, and e.g. sodium ion contained in the liquid may be removedusing an ion exchanger, but the desalting treatment may also beconducted by washing. Production costs can be reduced by desalting withthis washing step. From this desalting step as above described, atasteless and odorless collagen is obtained. The desalted treated liquidis, following a disinfecting treatment during a second disinfecting step35 e.g. by heating at 120° C. for 3 seconds, then cooled during a fifthcooling step 36 to e.g. 75° C. or less. The cooled treated liquid iscondensed during a condensing step 37, so that it reaches 40° Bx (±1.0°Bx) at an evaporation temperature of 60 to 65° C. By this treatment acollagen extract is obtained.

The collagen extract with water adjustment is added to theabove-described hydroxyapatite extract with water adjustment in theweight ratio of about 80% by weight of hydroxyapatite to about 20% byweight of collagen, both being calculated based on the solid content, sothat the mixture is stirred and mixed during a stirring and depositingstep 43. As at this point the collagen is deposited in thehydroxyapatite, the deposited and bonded product is subsequently driedduring a drying step 44, so that a solid hydroxyapatite/collagencomposite is ready. The composite is then crushed during a crushing step45 and further subjected to a mesh-passing step 46 during which thecomposite is mesh-passed, so that pulverized hydroxyapatite/collagencomposite can be obtained.

In the meantime, reference has been made in the foregoing to the casewhere steps for producing collagen extract are separated from those forproducing hydroxyapatite, however, the process for producing thecollagen is identical to that for hydroxyapatite up to a point wheredried fish scales restore the original water content and then aretreated by acid. To that point therefore the both processes undergoidentical steps, so that, needless to say, focus can also be placed onthe production line for the collagen within a overall production line,and in particular a production process as follows is also possible: thedried fish scales are allowed to restore their original water contentduring a soaking step 20 and then are treated by acid during an acidtreatment step 21, before hydroxyapatite extract is separated and thesubsequent steps concerning hydroxyapatite is separated for its ownproduction line, as shown in FIG. 2. The above-mentioned productionprocess is shown in FIG. 2, and steps with identical reference numeralsare identical to those in FIG. 1, therefore references thereto areomitted.

Thereafter, in obtaining a substance of fine-powdered hydroxyapatitefrom the above-mentioned hydroxyapatite extract, the extract is driedduring a drying step 12, in particular as shown in FIG. 1. The dryingstep 12 is not particularly limited provided that the hydroxyapatiteextract can be dried, but may be dried e.g. using hot blast. Atemperature of the hot blast is not particularly limited, but ispreferably 65 to 100° C., and more preferably 75° C. A duration fordrying is not particularly limited, but preferably 15 to 24 hours, andmore preferably 18 hours. After this step, dried fine-powderedhydroxyapatite can be obtained.

Subsequently, in obtaining a simple substance of fine-powdered collagenfrom the above-mentioned collagen extract, a condensed collagen extractis subjected to a drying step 48 wherein it is freeze-dried or otherwisedried using methods including spray drying so that dried fine-powderedcollagen can be obtained.

Fine-powdered collagen obtained in the above-described manner is atasteless, odorless, high-quality and low molecular weight collagen, ofa molecular weight of 500 to 1,000. Collagen contains a large amount ofamino acids, and its features vary depending on a composition of theseamino acids. In collagen the amino acid composition and sequence areunique in comparison to other proteins, in that three polypeptide chainsform clockwise helices respectively, thus keep a stable structure.Further a stable helical structure peculiar to collagen would not beformed without hydroxyproline, and therefore it can be safely statedthat the hydroxyproline content is reference value for determining thecollagen quality. In other words, collagen containing morehydroxyproline is better in quality. Results are listed in Table 1 froman analysis of a composition of the amino acids contained in thecollagen obtained by the process for producing collagen according to thepresent invention, using the automatic amino acid analysis. All theentries are contents in 100 g of collagen. In the meantime, amino acidsare also analyzed, as shown in Table 1, by the automatic amino acidanalysis concerning collagen derived from pork skin, collagen of CompanyA, and collagen of Company B.

TABLE 1 Amino Acid Composition of Collagen In grams Company PresentExample Pork Skin Company A B Gly 28.5 33.34 36.4 33.48 Ala 10.9 11.2811.1 12.36 Ser 3.59 3.33 5 3.84 Thr 2.65 1.83 2.3 2.83 1/2-cystine 0.030 0 0 Met 1.55 0.47 1.5 1.52 Val 2.48 2.18 1.7 2.06 Leu 2.66 2.49 2 2.14Ile 1.35 1.08 1.1 0.92 Phe 1.84 1.26 1.2 1.32 Tyr 0.37 0.35 0 0.29 Pro13.8 12.18 11 11.59 hydroxyproline 11.4 10 6 8 Lys 3.78 2.59 2.6 2.57His 0.58 0.5 0 0.5 Arg 8.58 4.7 5.1 5.16 Asp 6.15 4.32 5.5 4.14 Glu 10.44.57 7.8 6.97 Trp 0 0 0 0

As seen in Table 1 above, it is observed that collagen producedfollowing a process for producing contains more hydroxyproline thanthose produced using other materials and following other processes forproducing. Moreover, in this collagen reduction to low molecular weight(of a molecular weight of 500 to 1,000) is sought by an enzymaticdegradation, so that a biological absorption rate is high.

Subsequently, a powdery hydroxyapatite/collagen composite which isconstituent of a biocompatible material according to the presentinvention is produced as is described below. First concerning thehydroxyapatite extract, 2 kg of dried carp scales is placed into a nylonnet of an opening size of 1 mm or less. Then 6.6 kg of water, i.e. 3.3times as much as the dried fish scales, is poured into a PE(polyethylene) tank and the nylon net stuffed with the dried fish scalesis fully soaked in the water in the tank for 12 hours, so that the fishscales restore the original water content.

After the soaking step, water in a PE (polyethylene) tank is onceremoved and 6.6 kg of fresh water is again poured into the tank. Atemperature of the water in the tank is maintained at 20° C. (±2° C.),while 1,300 g of hydrochloric acid having purity 35% is added to thewater in order to obtain an aqueous hydrochloric acid solution. An acidtreatment is thus conducted by soaking and at the same time stirring thefish scales having the original water content in the aqueoushydrochloric acid solution for 25 minutes. A hydroxyapatite extractobtained by the acid treatment to the fish scales is subjected tosolid-liquid separation and then filtered using a filtering apparatus. Afiltered hydroxyapatite extract is deodorized using palm-chip activatedcarbon. After the deodorization treatment the treated liquid issubjected to clarifying filtration using a filter press, so that finesolids are removed from the treated liquid.

Sodium hydroxide (48% by weight NaOH) is added to the filteredhydroxyapatite extract and then the extract is stirred using stirrer sothat pH of the extract is adjusted to 7.

Hydroxyapatite is thus eluted by this treatment so that hydroxyapatiteparticles are deposited. After depositing, the solid is separated fromthe liquid using a filter press, and then the separated hydroxyapatiteis placed into a tank, to which 36 to 48 kg of water i.e. 3 to 4 timesas much as the treated liquid is added. The hydroxyapatite is thendesalted by stirring the liquid together with the water and thus washingit. Thereafter the liquid is heated to 70° C. and a disinfectingtreatment is maintained for 30 minutes, before the liquid is stirredusing a filter press for a solid-liquid separation. The hydroxyapatiteextract obtained in this manner contains 75% by weight of water and hasa weight of 1,800 g.

Subsequently, a collagen extract is produced as is described below. 150kg of dried carp scales is placed into a nylon net of an opening size of1 mm or less. Then, the nylon net stuffed with the dried fish scales isplaced into a PE (polyethylene) tank containing 495 kL of water, i.e.3.3 times as much as the dried fish scales and fully soaked in the waterin the PE tank for 12 hours, so that the fish scales restore theoriginal water content.

Next 495 kL of water, i.e. 3.3 times as much as the dried fish scales,is poured into another PE (polyethylene) tank and a temperature of thewater in the tank is maintained at 20° C. (±2° C.), while 93 kL ofhydrochloric acid having purity 35% is added to the water in order toobtain an aqueous hydrochloric acid solution. An acid treatment is thusconducted by soaking and at the same time stirring a nylon net stillcontaining the fish scales having the original water content in theaqueous hydrochloric acid solution for 15 minutes. The nylon netcontaining the acid-treated fish scales is then unfolded in a washingtank and subjected to 5 washing cycles for 5 minutes, each of thewashing cycles being conducted in 600 kL of water so as not to allow thefish scales inside the net to escape outside, so that pH of the fishscales is adjusted to 4.0 (±0.2).

Washed fish scales are then placed into an iron pot containing watereight times as much as the fish scales, and subjected to a first heatingtreatment which is conducted at 98° C. or higher for 3 hours. This firstheating treatment is conducted in order to adjust a final concentrationof the liquid at 10 to 12° Bx. If water is evaporated too much andremaining water is less than a desired amount, further water is added.In this case, marks like scales are preferably placed on the iron pot,so that addition of water can be conducted so as to place a watersurface each time at a reference mark.

After a first heating treatment a liquid is cooled to 75° C. or lower,and a solid is separated from the liquid in a liquid state after thefirst heating treatment, using a 2-stage vibrating sieve machinecomprising vibrating sieves of 20-200 mesh. Water four times as much asthe solid, i.e. fish scales, is added to the solid separated during thesolid-liquid separation and then the water containing the solid issubjected to a second heating treatment at 98° C. or higher for 2 hours.This second heating treatment is conducted in order to adjust a finalconcentration of the liquid at 10 to 12° Bx. If water is evaporated toomuch and remaining water is less than a desired amount, further water ispreferably added.

After a second heating treatment a liquid is cooled to 75° C. or lower,and a solid is separated from the liquid in a liquid state after thesecond heating treatment, using a 2-stage vibrating sieve machinecomprising vibrating sieves of 20-200 mesh. The liquid after the secondheating treatment, from which the solid is removed, undergoes adisinfecting treatment at 75° C. for 15 minutes, together with theliquid after a first heating treatment. After the disinfecting treatmentthe liquid is cooled to 60° C. and enzymatically degraded.

In the enzymatic degradation treatment pH of the heat-treated liquid isfirst adjusted to 6.5 (±0.2), and after that an alkaline enzyme derivedfrom Bacillus licheniformis section (Genencor Int., Inc., trade name:Protex 6L) is added to the substrate, 0.08% heat-treated liquid; theliquid containing the enzyme is then stirred at 60° C. (±1° C.) bystirrer for 8 hours.

After the enzymatic degradation treatment the enzymatically degradedliquid (treated liquid) is retained at 85° C. for 10 minutes so that theenzyme is inactivated, and then the treated liquid is cooled to 60° C.After cooling, the treated liquid is deodorized using palm-chipactivated carbon and then subjected to clarifying filtration using afilter press, so that the solid contained in the treated liquid isremoved. In the meantime, a third filtering step preferably includes adeodorizing treatment. The deodorizing treatment may be conducted e.g.using woody or palm-chip activated carbon. After the solid is removed,the treated liquid is desalted using an ion exchanger. After thedesalting treatment, the treated liquid is heated at 120° C. for 3seconds and thus subjected to a sterilizing treatment, and thereafter itis cooled to 75° C. or lower. The cooled treated liquid is thensubjected to a condensing treatment in a condensing tank at 60 to 65° C.in order to adjust to 40° Bx (±1.0° Bx). A collagen extract is obtainedin this manner.

Subsequently 1,800 g of hydroxyapatite extract is prepared, which isseparated from a solid during a third liquid-solid separation step 11among steps for producing hydroxyapatite as above described and contains75% by weight of water, while 220 g of low molecular weight collagenextract is prepared, which is obtained prior to a drying step within theabove-described steps for producing collagen and contains 50% by weightof water (the weight for each extract is calculated as solid weight).Thereafter these extracts are altogether placed into a treatment tank,and mixed and stirred in a room temperature for 10 to 20 minutes, sothat 2,020 g of deposited and bonded hydroxyapatite/collagen product isdeposited. When this deposited and bonded product is then dried under ahot blast atmosphere at 70° C. for 18 hours, 520.6 g ofhydroxyapatite/collagen composite with a water content of 5.8% isobtained. The product has a sickly ivory white color and a very weakodor.

In the present example 220 g of low molecular weight collagen extract isprepared, which is obtained within the above-described steps forproducing collagen, and more specifically prior to a drying step 48,wherein its water content is adjusted to 50% by weight, while 1,800 g ofhydroxyapatite extract is prepared, which is separated from a solidduring a third liquid-solid separation step 11 among steps for producinghydroxyapatite as above described, wherein its water content is adjustedto 75% by weight. Thereafter these extracts are altogether placed into atreatment tank, and mixed and stirred in a room temperature for 10 to 20minutes, so that 2,020 g of deposited and bonded hydroxyapatite/collagenproduct is deposited. When this deposited and bonded product is thendried during a subsequent drying step under a hot blast atmosphere at70° C. for 18 hours, 520.6 g of hydroxyapatite/collagen composite with awater content of 5.8% is obtained. The product has a sickly ivory whitecolor and a very weak odor.

When a composite obtained in the above-described manner which is thenscanned using a scanning electron microscope is compared to a marketedapatite/collagen mixture as scanned using a scanning electronmicroscope, exclusively amorphous objects are observed in the compositeA as shown in FIG. 4, while in the marketed mixture B two types ofobjects, i.e. spherical objects (collagen) and amorphous objects(hydroxyapatite) are both observed, wherein one type of objects aremerely adhered to another, as shown in FIG. 3. As is understood in theabove stated, in the composite A, two materials seem to be bonded toform a single material. When a single material consisting ofhydroxyapatite is scanned for reference, finer crystal grains ascompared to the composite A are clearly observed, so that one maysuppose that it comprises collagen which is bonded with hydroxyapatiteat gaps between crystallic structures.

From a further analysis of the above-mentioned composite, results shownin Table 2 are obtained.

TABLE 2 LOT: 61201M Analyzed and Tested Item Result Note Method WaterContent 4.2 g/100 g Heat Drying Method at Atmospheric Pressure Collagen20.4 g/100 g 1 Kjeldahl Method Sodium 390 mg/100 g Atomic AbsorptionSpectroscopy Phosphorus 14.2 g/100 g ICP-Emission SpectrometryHydroxyapatite 70.2 g/100 g 2 ICP-Emission Spectrometry Magnesium 323mg/100 g ICP-Emission Spectrometry pH 6.6 3 Glass Electrode MethodNote 1. nitrogen-protein conversion factor: 6.25 Note 2. conversionfactor from calcium: 2.5067 Note 3. measured based on 10% suspension

As is seen in Table 2 above, the composite contains, as per 100 g, 4.2 gof water content (as measured by heat drying method at atmosphericpressure), 20.4 g of collagen (as measured by Kjeldahl method(nitrogen-protein conversion factor—6.25)), 14.2 g of phosphorus (asmeasured by ICP-emission spectrometry), 70.2 g of hydroxyapatite (asmeasured by ICP-emission spectrometry (conversion factor fromcalcium—2.5067)), 390 mg of sodium (as measured by atomic absorptionspectroscopy), 323 mg of magnesium (as measured by ICP-emissionspectrometry), and pH of the composite is 6.6 (as measured by glasselectrode method (measured based on 10% suspension)). In the meantime,28.0 g of calcium is identified by means of ICP-emission spectrometry in70.2 g of hydroxyapatite. The calculation formula for determining thecalcium content, 28.0 g, is as follows:

Ca₅(PO₄)₃OH = 5 × 40.078 + 3 × (30.973762 + 4 × 15.9994) + 15.9994 + 1.00794 = 502.311426Conversion Factor from Calcium

Ca₅(PO₄)₃OH/(5 × Ca) = 502.311426/(5 × 40.078) = 2.506669125 → 2.506770.2  g/2.5067 ≈ 28.0  g

Moreover product standards for the composite are preferably those shownin Table 3 below. According to Table 3, it contains 10% or less of water(as measured by heat drying method at atmospheric pressure), 15% or moreof collagen (as measured by Kjeldahl method (nitrogen-protein conversionfactor—5.55)), 10% or more of phosphorus (as measured by ICP-emissionemission spectrometry), 65% or more of hydroxyapatite (as measured byICP-emission emission spectrometry (conversion factor fromcalcium—2.5067)), 0.5% or less of sodium (as measured by atomicabsorption spectroscopy), 20 ppm or less of heavy metal (in form of Pb)(as measured by sodium sulfide calorimetric method), 2 ppm or less ofarsenic (in form of As₂O₃) (as measured by atomic absorptionspectroscopy), and of the composite is 6.0 to 8.0 (as measured by glasselectrode method (measured based on 10% solution)), common bacteriacount (viable cell count) is 3000 cells/g or less (as measured bystandard agar culture method), coliform organisms are negative (asmeasured by BGLB method).

TABLE 3 Quality Standards Item Standard Value Test Method Water Content10% or less Heat Drying Method at Atmospheric Pressure Collagen 15% ormore Kjeldahl Method (Note 1) Phosphorus 10% or more ICP-EmissionSpectrometry Hydroxyapatite 65% or more ICP-Emission Spectrometry (Note2) Sodium 0.5% or less Atomic Absorption Spectroscopy pH 6.0 to 8.0Glass Electrode Method (Note 3) Heavy Metal (in form of 20 ppm or lessSodium Sulfide Pb) Colorimetric Method Arsenic (in form of As₂O₃) 2 ppmor less Atomic Absorption Spectroscopy Common Bacteria Count 3000cells/g or Standard Agar Culture (Viable Cell Count) less MethodColiform Organisms Negative BGLB Method (Note 1) nitrogen-proteinconversion factor: 5.55 (Note 2) conversion factor from calcium: 2.5067(Note 3) measured based on 10% solution

Moreover, a study has been made for the effects of the compositeobtained in the above-described manner on the bone improvements, in caseof administration to osteoporotic model mice.

For study ICR female model mice of 10 weeks (of 20 g) were used. Thesemice underwent ovariectomy (OVX control group) and sham operation (shamoperated group), and were subsequently fed with a conventional diet for2 months, and then divided into 6 groups, each group having 10 mice. Ofthese, an OVX control group and a sham operated group were fed with 1.5to 1.8% calcium containing feed which contained calcium carbonate as acalcium source. Further as a reference group, one group was treated witha bisphosphonate preparation (risedronate), which is ananti-osteoporotic agent showing obviously higher bone density. Remaining3 groups were fed with special feeds, containing foodstuff and foodadditives. Combinations of foodstuff and food additives used in thepresent study are shown below in Table 4.

TABLE 4 Drug Content 1 sham None 2 OVX control None 3 risedronate 10μg/10 μL/day at one dose 4 Vitamin K₂ (MK-4) 500 μg/100 g of feed 5Vitamin K₂ (MK-7) 500 μg/100 g of feed 6 Composite 2 g of Ca/100 g offeed

After being fed these feeds for 2 months, mice were decapitated forblood collection. Then serum was separated from the collected blood, andafter that calcium, phosphorus, and magnesium concentrations andalkaline phosphatase level of the serum were determined. Furthermore,after a resection of femurs, a three-dimensional bone densitometry andbone structure analysis were conducted on the femoral distal epiphysesand diaphyses, using pQCT (Peripheral Quantitative Computed Tomography)and μCT (micro focus X-ray CT).

For an analysis of the trabecular structure, three-dimensional imagedata of femoral distal epiphyses were acquired using X-ray μCT(MCT-CB130F, Hitachi Medico), of 8×8 μm focus, with micro focus X-raytube. As conditions for imaging, tube voltage was 40 kV, tube current100 μA, and voxel size 17.8×17.8×17.8 μm, so that 50 volume data foreach were acquired. Three-dimensional images were reconstructed based onthe acquired 50 two-dimensional images of a trabecular bone and acortical bone. 3D parameters were then determined on these 3D imagesusing trabecular structure measuring software (TRI/3D-BON; RATOC SystemEngineering, Co., Ltd). The determined structural parameters are listedbelow in Table 5.

TABLE 5 BS/BV (1/mm) Bone Surface Area BV/BT (%) Bone Mass per TissueVolume (TV) Tb.Th (mcm) Trabecular Width Tb.N (1/mm) Trabecular NumberTb.Sp. (mcm) Trabecular Separation Tb.Spac (mcm) Trabecular Spacing DComplexity TBPf (1/mm) Trabecular Bone Pattern Factor SMI StructureModel Index V*m.space Marrow Space Star Volume V*tr Trabecular StarVolume N.Nd Number of Nodes N.Tm Number of Terminus N.Ct Number ofCortical Bones N.Nd/TV (1/mm3) Nodes per Tissue Volume (TV) N.Tm/TV(1/mm3) Terminus per Tissue Volume (TV) N.Ct/TV Cortical Bones perTissue Volume (TV) TSL Total Trabecular Skeletal Length NdNd/TSL (%)Ratio of Node-to-Node Strut Length to Total Trabecular Skeletal LengthCtNd/TSL (%) Ratio of Cortical Bone-to-Node Strut Length to TotalTrabecular Skeletal Length CtCt/TSL (%) Ratio of CorticalBone-to-Cortical Bone Strut Length to Total Trabecular Skeletal LengthTmTm/TSL (%) Ratio of Terminus-to-Terminus Strut Length to TotalTrabecular Skeletal Length TSL/TV (1/mm2) Total Trabecular SkeletalLength per Tissue Volume (TV) NdNd/TV (1/mm2) Node-to-Node Strut Lengthper Tissue Volume (TV) CtNd/TV (1/mm2) Cortical Bone-to-Node StrutLength per Tissue Volume (TV) CtCt/TV Cortical Bone-to-Cortical BoneStrut Length per Tissue Volume (TV) TmTm/TV (1/mm2) Terminus-to-TerminusStrut Length per Tissue Volume (TV)

Student's t-test was used for statistical treatment for comparisonbetween the groups. Only the parameters showing significant differencein the OVX control group are compared to those in the remaining groups.

Subsequently, a bone mass density (mg/cm³) as a physical quantity wasanalyzed separately for trabecular bones and cortical bones based onthree-dimensional bone densitometry conducted using pQCT on the femoraldistal epiphyses and diaphyses and cross-sectional images acquired inpQCT, and bone strength index was identified based on cortical bonewidth, second moment of area, and section modulus etc. Analyzedparameters are listed in Table 6. Student's t-test was used forstatistical treatment for comparison between the groups. Sitesdesignated for analysis are shown in FIG. 6.

TABLE 6 ANALYZED PARAMETERS Epiphyses Bone Density (mg/cm3) TrabecularBone Density (mg/cm3) Cortical Bone Density (mg/cm3) Cortical BoneCross-Section (mm2) Cortical Bone Thickness (mm) PeriostealCircumference (mm) Bone Strength Index (mm3) Diaphyses Bone Density(mg/cm3) Trabecular Bone Density (mg/cm3) Cortical Bone Density (mg/cm3)Cortical Bone Cross-Section (mm2) Cortical Bone Thickness (mm)Periosteal Circumference (mm) Bone Strength Index (mm3)

Subsequently, blood was collected from the tested mice of each group,and mineral concentration and ALP activity in serum were determined.Blood collection was conducted by decapitating under a twilight sleep.The collected blood stood for 1 hour at ice temperature, and thereafterthe serum was separated by a centrifuge for 10 minutes at 1000 rpm.Calcium amount was determined by chelete coloring method. 50 μL of serumwas added to 5 mL of a 0.88 M monoethanolamine buffer adjusted to pH=11,and a mixture of o-cresol-phthalein complexone and 8-quinolinol as acoloring agent was further added. The resulting mixture was sufficientlystirred and stood for 5 minutes, and thereafter absorbance at 570 nmdetermined using a spectrometer. Phosphorus was determined by directmolybdenum blue method. After 0.2 mL of serum was added to 5 mL of acoloring agent and the resulting mixture stood for 15 minutes at roomtemperature, absorbance at 690 nm was determined using a spectrometer.The coloring agent was prepared by adding a surfactant to a mixture ofammonium molybdate, ammonium ferrous sulfate and sulfate. Magnesium wasdetermined by xylidyl blue method. 3 mL of a coloring agent was added to20 μL of serum and the resulting mixture stood for 10 minutes at roomtemperature, and thereafter absorbance at 520 nm was determined using aspectrometer. The coloring agent was prepared by adding a surfactant toa mixture of xylylazo violet I and ethylene glycol tetraacetic acid.

Alkaline phosphatase was determined by phenyl phosphoric acid substratemethod. After 50 μL of serum was added to 2 mL of a substrate buffer andheated at 37° C. for 15 minutes, 2 mL of 36 mM potassium ferricyanide.The resulting mixture was sufficiently stirred, and thereafterabsorbance at 570 nm was determined using a spectrometer. The substratebuffer was prepared by adding a mixture of 4-aminoantipyrine andphenylphosphoric acid to 50 mM carbonate buffer adjusted to pH=10.5.

In all of these determinations assay kits for clinical and biochemicaluse manufactured by Wako Pure Chemicals, Co., Ltd. were used.

TABLE 7 MINERAL CONCENTRATION AND ALP ACTIVITY IN MOUSE SERA Mg ALP Ca(mg/dL) P (mg/dL) (mg/dL) (BL unit) 1. sham 9.13 ± 1.02 7.02 ± 0.72 3.10± 1.05 3.88 ± 1.54 2. OVX 9.00 ± 0.32 7.01 ± 0.78 2.72 ± 0.72 4.26 ±1.98 3. Risedronate 9.02 ± 1.15 6.95 ± 0.32 3.12 ± 1.21 3.65 ± 1.26 4.MK-4 9.60 ± 0.51 6.96 ± 0.44 2.67 ± 0.28 3.52 ± 1.82 5. MK-7 9.34 ± 1.507.12 ± 0.64 2.63 ± 0.63 3.77 ± 1.76 6. Composite 9.80 ± 1.64 7.06 ± 0.762.71 ± 0.92 3.44 ± 1.59

Table 7 above shows mineral concentrations and alkaline phosphatase(ALP) activity in model mouse sera. As per blood Ca, P and Mg, nosignificant difference could not be observed between all the testedgroups. On the other hand, concerning ALP activity which shows high ratein presence of bone metabolic abnormalities, no significant differencewas observed in the present test results, although all groups tended toshow low rates as compared to the OVX control group.

Table 8 below shows a significant difference comparison between a OVXcontrol group and tested groups in femoral distal epiphyses and femoraldistal diaphyses which were measured by pQCT. In the OVX control groupand a sham operated group 11 parameters showed a significant difference,which demonstrated that the OVX control group obviously consisted ofosteoporotic model mice. Moreover, a group treated with risedronatewhich is an anti-osteoporotic agent showed a significant increase inbone density, trabecular bone density, cortical bone thickness andcortical bone cross-section of the epiphyses, and in cortical bonethickness and cortical bone cross-section of the diaphyses, as comparedto the OVX control group. In this manner an effect of risedronatecharacterized in a bone density increase was proven, which demonstratedthat the present test was conducted with high precision. Further, amongthe tested groups, a composite group showed the most similar effect tothe group treated with risedronate. In the meantime, MK7 showed a moresignificant increase in cortical bone thickness, cortical bonecross-section, periosteal circumference and bone strength index of thefemoral distal epiphyses, as compared to the OVX control group. However,in the diaphyses, no significant difference is observed as compared tothe OVX control group. In addition, in MK4 group only an epiphysealperiosteal circumference significantly increased as compared to the OVXcontrol group.

TABLE 8 OVX sham Risedronate Composite MK4 MK7 Epiphyses Bone Density(mg/cm3) p < 0.01 p < 0.01 Trabecular Bone Density p < 0.05 p < 0.05(mg/cm3) Cortical Bone Density p < 0.01 p < 0.01 (mg/cm3) Cortical BoneCross-Section p < 0.01 p < 0.05 p < 0.05 p < 0.05 (mm2) Cortical BoneThickness (mm) p < 0.01 p < 0.05 p < 0.05 p < 0.05 PeriostealCircumference (mm) p < 0.05 p < 0.01 Bone Strength Index (mm3) p < 0.05p < 0.01 p < 0.05 Diaphyses Bone Density (mg/cm3) p < 0.01 TrabecularBone Density p < 0.05 p < 0.05 p < 0.05 (mg/cm3) Cortical Bone Density p< 0.01 p < 0.05 (mg/cm3) Cortical Bone Cross-Section p < 0.01 p < 0.05(mm2) Cortical Bone Thickness (mm) p < 0.01 p < 0.05 PeriostealCircumference (mm) Bone Strength Index (mm3) Items showing a significantdifference 11 6 6 2 4

FIG. 7 shows representative pQCT images of an OVX control group and acomposite group, on femoral distal epiphyses (A) and diaphyses (B) ofmodel mouse. An area in white represents a cortical bone, while areasinside and outside is trabecular bones. The cortical bone had morethickness in a mouse treated with the composite, which enabled tovisually confirm a rise in bone density.

Table 9 below shows results of a three-dimensional structure analysis ofbone internal structure of model mouse using μCT in femoral distalepiphyses. Parameters of the tested groups in the bold-bordered cellsshowed higher means than those of the OVX control group, whichdemonstrated better effects on improvement of bone internal structure.On the other hand, parameters in the cells without bold borders showedlower means than those of the OVX control group, and the lower themeans, the better the effects on improvement of bone internal structure.In other words, if Tb.SP, TB space, TBPf, VmSpace, N.Tm, TmTm/TSL,TmTm/TV show a lower value, they show better effects on improvement ofbone internal structure. A sham operated group showed significanteffects on improvement of trabecular bone structure concerning 11parameters, while a risedronate group concerning 12, which demonstratedthat the present experimental system was implemented with highprecision. After these, MK7, the composite and MK4 is arranged in thisorder according to the importance of the effects on improvement oftrabecular bone structure.

TABLE 9

FIG. 8 shows 3-D restructured images of representative trabecularstructure of an OVX control group and a sham operated group intransverse and longitudinal cross-sections. FIG. 9 equally shows 3-Drestructured images of representative trabecular structure of arisedronate group, a MK4 group, a MK7 group and a composite group intransverse and longitudinal cross-sections.

As described in detail in the foregoing, as a result of the 3-D imageanalysis conducted using OVX mice in distal epiphyses and diaphyses foreffects on an improvement of bone density and trabecular structure,based on images shown in FIGS. 8 and 9, the following conclusions weredrawn:

-   1. The composite showed both effects on an increased bone density    and improved trabecular structure, with the increase in bone density    being a prevalent effect. Moreover, merely the composite showed an    increase in diaphyseal cortical bone density.-   2. MK7 showed both effects on an increased bone density and improved    trabecular structure, with the improvement in trabecular structure    being prevalent effect.-   3. For MK4 an improvement in bone internal structure was observed as    a prevalent effect.

From the effects listed above it is evident that the composite shows amarked effect on an increased bone density, with an improvement in boneinternal structure being further expected, thus the composite iseffective in prevention for osteoporosis and improvement for adeteriorated bone structure.

As described in the forgoing a hydroxyapatite/collagen composite forminga biocompatible material according to the present invention has almostno odor and taste, proves safe as it is made of fish scales, and has anexcellent in effect on bone quality improvement. Therefore, it is clearthat the composite is useful to improve and prevent various diseases andsymptoms due to calcium deficiency including osteoporosis, by taking itas is or mixed with other foodstuff, confectionery, chewing gum,drinking water, etc. Moreover, the process for producing the compositecan be incorporated into those for producing a fish scale-derivedhydroxyapatite or collagen, which enables an easy manufacture.

1. A biocompatible material containing a composite of hydroxyapatite andcollagen, wherein the biocompatible material is manufactured by dryingthrough a hot blast atmosphere a mixture of (i) a fish scale-extractedhydroxyapatite having a water content of 70 to 75% by weight and (ii) afish scale-extracted collagen having a water content of 40 to 60% byweight, and wherein the dried biocompatible material has a weightpercent ratio of hydroxyapatite to collagen of about 8:2.
 2. Thebiocompatible material according to claim 1, wherein said collagencontains 11 g or more of hydroxyproline per 100 g thereof.
 3. Thebiocompatible material according to claim 1, wherein said materialcontains by weight (w/w) 10% or less water, 15% or more of collagen, 65%or more of hydroxyapatite said hydroxyapatite comprising 10% or more ofphosphorus, and 0.5% or less of sodium.
 4. The biocompatible materialaccording to claim 1, wherein said composite contains 4.2 g of water;20.4 g of collagen; 70.2 g of hydroxyapatite said hydroxyapatitecomprising 14.2 g of phosphorus; 390 mg of sodium; and 323 mg ofmagnesium per 100 g.
 5. The biocompatible material according to claim 1,wherein said hydroxyapatite has a molecular weight of 1,000 Da.
 6. Thebiocompatible material according to claim 1, wherein said collagen has amolecular weight of 500 to 1,000 Da.
 7. A process for producing abiocompatible material containing a composite of hydroxyapatite andcollagen, the process comprising the steps of: mixing a fishscale-obtained hydroxyapatite extract said hydroxyapatite extract havinga water content by weight thereof of 70 to 75% (w/w) with a fishscale-obtained collagen extract said collagen extract having a watercontent by weight thereof of 40 to 60% (w/w) in ahydroxyapatite:collagen weight percent ratio of about 8:2; stirring themixture; and drying the stirred mixture by hot blast thereby obtainingthe biocompatible material containing said composite.
 8. The process forproducing the biocompatible material according to claim 7, furthercomprising mixing into a food or feed material an amount of the driedbiocompatible material sufficient to improve a calcium deficiencysymptom of trabecular or cortical bone the symptom improvement selectedfrom the group consisting of increasing bone thickness and increasingbone density.